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Genomic characterization of carbapenemase-producing Enterobacterales from wastewater reveals the convergence of KPC-2 and GES-16 in Brazil.

Carbapenemase-producing Enterobacterales (CPE) pose a significant public health concern due to the limited therapeutic options and increasing dissemination outside clinical settings. Wastewater treatment plants (WWTPs) have been proposed as relevant environmental reservoirs for antimicrobial-resistant bacteria and mobile genetic elements. In this study, we performed genomic characterization of CPE strains recovered from raw wastewater samples from the influents of different WWTPs. Antimicrobial susceptibility testing revealed multidrug resistance, including coresistance to carbapenems and polymyxins, among Klebsiella pneumoniae, Enterobacter asburiae, and Enterobacter kobei strains. Whole-genome sequencing identified the convergence of the blaKPC-2 and blaGES-16 genes, as well as the presence of blaGES-5 in E. kobei sequence type (ST) 540 strains. Moreover, the blaKPC-2 gene was detected in K. pneumoniae strains belonging to high-risk clones ST11 (capsular types KL64 and KL15) and ST307 (capsular type KL102), and E. asburiae ST384. The blaGES-5 and blaGES-16 genes were associated with class 1 integrons, while the blaKPC-2 gene was embedded within transposons (Tn4401a, Tn4401i, and Tn3-like) and insertion sequences (ISKpn27 and ISKpn6). Notably, genomic analyses and literature review demonstrated that the blaGES-16 gene remains unique to Brazil. The putative pathogenic potential of carbapenem-resistant K. pneumoniae ST11 was also assessed. These findings support the environmental circulation of clinically relevant Enterobacterales genotypes and emphasize the potential role of WWTPs as conduits for CPE dissemination, if not adequately operated. Therefore, genomic surveillance in extra-hospital settings may contribute to a better understanding of antimicrobial resistance ecology and inform One Health mitigation strategies.

Brazil↗

Rat L (long interspersed repeated DNA) elements contain guanine-rich homopurine sequences that induce unpairing of contiguous duplex DNA.

The L family (long interspersed repeated DNA) of mobile genetic elements is a persistent feature of the mammalian genome. In rats, this family contains approximately equal to 40,000 members and accounts for approximately equal to 10% of the haploid genome. We demonstrate here that the guanine-rich homopurine stretches located at the right end of L-DNA induce oligonucleotide uptake by contiguous duplex DNA. The uptake is dependent on negative supercoiling and the length of the homopurine stretch and occurs even when the L-DNA homopurine stretches are introduced into a different DNA environment. The bound oligomer primes DNA synthesis when DNA polymerase and deoxyribonucleoside triphosphates are added, resulting in a faithful copy of the template to which the oligonucleotide had bound. The implications of this property of the L-DNA guanine-rich homopurine stretches in the amplification, recombination, and dispersal of L elements is discussed.

Animals↗

Mobilized mercury in subjects with varying exposure to elemental mercury vapour.

In a mercury mobilization test, 0.3 g of the complexing agent sodium 2,3-dimercaptopropane-1-sulfonate (DMPS) was given orally to 10 workers with moderate occupational exposure to elemental mercury vapour, to 8 dentists with slight exposure, to 18 matched controls, and to 5 referents without amalgam fillings. In the workers, DMPS caused an increase in 24-h urinary mercury excretion by a factor of 10; in the dentists, 5.9; in the controls, 5.3; and in the amalgam-free referents, 3.8. Of the mercury excreted during 24 h, 59% appeared during the first 6 h. Close, albeit non-linear, associations were found between mobilized mercury and the premobilization mercury levels in plasma and urine, but not with the duration of occupational exposure or the rough estimate of the integrated function of blood levels vs time. The present data indicate that mercury mobilized after a single DMPS dose in close connection with exposure is mainly an index of recent exposure and is not significantly affected by slow body pools or long-term exposure.

Adult↗

Comparison of SXT and R391, two conjugative integrating elements: definition of a genetic backbone for the mobilization of resistance determinants.

The SXT element (SXT) is becoming an increasingly prevalent vector for the dissemination of antibiotic resistances in Vibrio cholerae. SXT is a member of a larger family of elements, formerly defined as IncJ plasmids, that are self-transmissible by conjugation and integrate site-specifically into the host chromosome. Comparison of the DNA sequences of SXT and R391, an IncJ element from Providencia rettgeri, indicate that these elements consist of a conserved backbone that mediates the regulation, excision/integration and conjugative transfer of the elements. Both elements have insertions into this backbone that either confer the element-specific properties or are of unknown function. Interestingly, the conserved SXT and R391 backbone apparently contains hotspots for insertion of additional DNA sequences. This backbone represents a scaffold for the mobilization of genetic material between a wide range of gram-negative bacteria, allowing for rapid adaptation to changing environments.

Base Sequence↗

RNAs from all categories generate retrosequences that may be exapted as novel genes or regulatory elements.

While the significance of middle repetitive elements had been neglected for a long time, there are again tendencies to ascribe most members of a given middle repetitive sequence family a functional role--as if the discussion of SINE (short interspersed repetitive elements) function only can occupy extreme positions. In this article, I argue that differences between the various classes of retrosequences concern mainly their copy numbers. Consequently, the function of SINEs should be viewed as pragmatic such as, for example, mRNA-derived retrosequences, without underestimating the impact of retroposition for generation of novel protein coding genes or parts thereof (exon shuffling by retroposition) and in particular of SINEs (and retroelements) in modulating genes and their expression. Rapid genomic change by accumulating retrosequences may even facilitate speciation [McDonald, J.F., 1995. Transposable elements: possible catalysts of organismic evolution. Trends Ecol. Evol. 10, 123-126.] In addition to providing mobile regulatory elements, small RNA-derived retrosequences including SINEs can, in analogy to mRNA-derived retrosequences, also give rise to novel small RNA genes. Perhaps not representative for all SINE/master gene relationships, we gained significant knowledge by studying the small neuronal non-messenger RNAs, namely BC1 RNA in rodents and BC200 RNA in primates. BC1 is the first identified master gene generating a subclass of ID repetitive elements, and BC200 is the only known Alu element (monomeric) that was exapted as a novel small RNA encoding gene.

Animals↗

Negative regulation of chemokine receptor CXCR4 by tumor suppressor p53 in breast cancer cells: implications of p53 mutation or isoform expression on breast cancer cell invasion.

Chemokine receptor CXCR4 and its ligand CXCL12 are suggested to be involved in migration, invasion and metastasis of breast cancer cells. Mutation of the tumor suppressor gene p53 in breast cancer is associated with metastasis and aggressive clinical phenotype. In this report, we demonstrate that wild type but not the dominant-negative mutant (V143A) or cancer-specific mutants (R175H or R280K) of p53 repress CXCR4 expression. Recently described cancer-specific p53 isoform, Delta133p53, also failed to repress CXCR4 promoter activity. Short-interfering RNA-mediated depletion of p53 increased endogenous CXCR4 expression in MCF-7 breast cancer cells that contain wild-type p53. Basal CXCR4 promoter activity in HCT116 colon carcinoma cells deleted of p53 [HCT116(p53KO)] was 10-fold higher compared to that in parental HCT116 cells with functional wild-type p53. Deletion analysis of CXCR4 promoter identified a seven-base pair p53-repressor element homologous to cyclic AMP/AP-1 response (CRE/AP-1) element. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed binding of ATF-1 and cJun to the CRE/AP-1 element. The p53 rescue drug PRIMA-1 reduced CXCR4 mRNA and cell surface expression in MDA-MB-231 cells, which express R280K mutant p53. CP-31398, another p53 rescue drug, similarly reduced cell surface levels of CXCR4. PRIMA-1-mediated decrease in CXCR4 expression correlated with reduced invasion of MDA-MB-231 cells through matrigel. These results suggest a mechanism for elevated CXCR4 expression and metastasis of breast cancers with p53 mutations or isoform expression. We propose that p53 rescue drugs either alone or in combination with chemotherapeutic drugs may be effective in reducing CXCR4-mediated metastasis.

Breast Neoplasms↗

Feedback on hypothalamic TRH transcription is dependent on thyroid hormone receptor N terminus.

The beta thyroid hormone receptor (TRbeta), but not TRalpha1, plays a specific role in mediating T(3)-dependent repression of hypothalamic TRH transcription. To investigate the structural basis of isoform specificity, we compared the transcriptional regulation and DNA binding obtained with chimeric and N-terminally deleted TRs. Using in vivo transfection assays to follow hypothalamic TRH transcription in the mouse brain, we found that TRbeta1 and chimeras with the TRbeta1 N terminus did not affect either transcriptional activation or repression from the rat TRH promoter, whereas N-terminally deleted TRbeta1 impaired T(3)-dependent repression. TRalpha1 or chimeras with the TRalpha1 N terminus reduced T(3)-independent transcriptional activation and blocked T(3)-dependent repression of transcription. Full deletion of the TRalpha1 N terminus restored ligand-independent activation of transcription. No TR isoform specificity was seen after transcription from a positive thyroid hormone response element. Gel mobility assays showed that all TRs tested bound specifically to the main negative thyroid hormone response element in the TRH promoter (site 4). Addition of neither steroid receptor coactivator 1 nor nuclear extracts from the hypothalamic paraventricular nuclei revealed any TR isoform specificity in binding to site 4. Thus N-terminal sequences specify TR T(3)-dependent repression of TRH transcription but not DNA recognition, emphasizing as yet unknown neuron-specific contributions to protein-promoter interactions in vivo.

Animals↗

Evaluation of the critical factors controlling stability of chromium, copper, arsenic and zinc in iron-treated soil.

Various environmental factors are expected to affect the mobility of elements in chemically stabilized soils. The aim of this study was to evaluate the effects of pH, oxidizing-reducing potential (Eh), liquid-to-solid ratio (L/S), presence of organic matter (OM) and microbial activity (MA) on the mobility of chromium, copper, arsenic and zinc in zerovalent iron (Fe(0))-stabilized soil. A 2(5) full factorial design was applied to assess the leaching of the elements from the treated soil. The factor having the most impact on the mobility of Cr, Cu and Zn was pH; low pH (3) led to the release of these elements. Arsenic remobilization was controlled by L/S and MA, whilst Eh, though also significant, had less influence. In the identified worst-case scenarios, more than half of the total Zn and Cu and 14% of As can be expected to remobilize from the treated soil. The leaching procedure concerning sample agitation and type of filtration showed to substantially affect the results of As leaching, especially in OM rich soil.

Arsenic↗

Genetic definition of a protein-splicing domain: functional mini-inteins support structure predictions and a model for intein evolution.

Inteins are protein-splicing elements, most of which contain conserved sequence blocks that define a family of homing endonucleases. Like group I introns that encode such endonucleases, inteins are mobile genetic elements. Recent crystallography and computer modeling studies suggest that inteins consist of two structural domains that correspond to the endonuclease and the protein-splicing elements. To determine whether the bipartite structure of inteins is mirrored by the functional independence of the protein-splicing domain, the entire endonuclease component was deleted from the Mycobacterium tuberculosis recA intein. Guided by computer modeling studies, and taking advantage of genetic systems designed to monitor intein function, the 440-aa Mtu recA intein was reduced to a functional mini-intein of 137 aa. The accuracy of splicing of several mini-inteins was verified. This work not only substantiates structure predictions for intein function but also supports the hypothesis that, like group I introns, mobile inteins arose by an endonuclease gene invading a sequence encoding a small, functional splicing element.

Amino Acid Sequence↗

Insertional mutagenesis of the myc locus by a LINE-1 sequence in a human breast carcinoma.

The proto-oncogene c-myc is the cellular homologue of the transforming sequence carried by the avian myelocytomastosis virus MC29. A growing body of evidence implicates structural and functional alterations in and around proto-oncogenes such as c-myc in tumorogenesis. Here we report that comparison of the structure of myc from a ductal adenocarcinoma of the breast and from normal breast tissue of the same patient (Sc) revealed a tumour-specific rearrangement of one myc locus and amplification of the other myc locus. (For myc reviews see refs 1-4; for myc involvement in breast neoplasia see refs 5-7.) Within the second intron of the rearranged locus was a non-myc sequence with nearly complete homology to a long interspersed repetitive element (a LINE-1 sequence or L1). In this case, the L1 sequence has functioned as a mobile genetic element to produce a somatic mutation.

Base Sequence↗

Cloning and characterization of the promoter region of the human CD83 gene.

Human CD83 (hCD83) is a glycoprotein expressed predominantly on the surface of dendritic cells (DC). To get insight into the regulation of hCD83 expression, we cloned a 3037 bp fragment up-stream of the translation initiation codon. Deletion mutants were constructed revealing highest promoter activity in the -261 fragment containing four SP1 binding sites and one NF-kappaB element. Electrophoretic mobility shift assays demonstrated the specific interaction of NF-kappaB factors with the NF-kappaB element as well as specific binding of SP1 and SP3 to the SP1 binding site. The hCD83 promoter was inducible by TNF-alpha. This inducibility was strictly dependent on the intact NF-kappaB element.

Antigens, CD↗

The characterization of terminators of RNA transcription on IS30 and an analysis of their role in IS element-mediated polarity.

Using expression vectors carrying the lacUV5 or Pgal promoters and the galK gene, we have studied terminators of transcription on the prokaryotic mobile genetic element IS30. The long open reading frame, ORF-A, of IS30 contains a relatively Rho-independent terminator, T30A, within its coding sequence. T30A terminates the majority of transcripts initiated at either an external promoter or the IS30-borne promoter P30A. No other terminator functions on this strand of IS30 (orientation left to right). In the orientation right to left, the previously identified terminator T30C, which follows ORF-C, is Rho-independent. T30C together with T30D, a newly identified, strong, partially Rho-dependent terminator near the left end of IS30, permits less than 2% read-through from external promoters. Neither ORF-A nor ORF-C appears to be protected from transcription by external promoters. As a consequence of the internal terminators, the insertion of IS30 into an operon can be expected to reduce the expression of genes downstream of the site of insertion weakly for one orientation of IS30 and strongly for the other orientation.

Base Sequence↗

A temperature-sensitive IE1 protein of Autographa californica nuclear polyhedrosis virus has altered transactivation and DNA binding activities.

The temperature-sensitive mutant tsB821 of Autographa californica nuclear polyhedrosis virus has two nucleotide transitions in the transregulatory ie1 gene (Ribiero et al. (1994) J. Virol. 68, 1075-1084). These mutations result in two conservative amino acid substitutions in the encoded protein. To determine whether the mutations affect the ability of IE1 to interact with viral enhancer elements, electrophoretic mobility shift assays were performed using extracts prepared from infected cells and a fragment of the viral enhancer element hr5. When binding reactions were assembled on ice, the extracts prepared from tsB821-infected cells showed wild-type levels of DNA binding activity, whether the infected cells were grown at the permissive or nonpermissive temperature. However, when binding reactions were incubated at 33 degrees, enhancer binding activity was significantly reduced in the ts extract. This suggests that the ts phenotype results from reduced interactions of tsIE1 with the viral enhancer elements. Site-directed mutagenesis was used to construct plasmids encoding IE1 with both of the amino acid substitutions (tsIE1) and with each of the single substitutions. tsIE1 was expressed in transient assays and exhibited thermolabile enhancer binding activity. However, proteins with single substitutions did not accumulate to detectable levels and did not exhibit enhancer binding activity. Temperature shift-up experiments with tsB821 indicated that late genes were expressed at wt levels when cells were shifted to the nonpermissive temperature during the early phase. This suggests that IE1 is not directly and continuously required for expression of late genes. However, polyhedrin expression was decreased in the mutant-infected cells when the temperature was shifted during the early or late phases of infection. Together, these results suggest that IE1 may differentially affect expression of these two classes of genes.

Animals↗

Interleukin-1beta suppresses retinoid transactivation of two hepatic transporter genes involved in bile formation.

Cytokines have been implicated in the pathogenesis of inflammatory cholestasis. This is due to transcriptional down-regulation of hepatic transporters including the Na(+)/bile acid cotransporter, ntcp, and the multispecific organic anion exporter, mrp2. We have recently shown that ntcp suppression by lipopolysaccharide in vivo is caused by down-regulation of transactivators including the previously uncharacterized Footprint B-binding protein. Both the ntcp FpB element and the mrp2 promoter contain potential retinoid-response elements. We hypothesized that retinoic acid receptor (RAR) and retinoid X receptor (RXR) heterodimers would activate these two genes and that cytokines that reduce bile flow might do so by suppressing nuclear levels of these transactivators. Retinoid transactivation and interleukin-1beta down-regulation of the ntcp and mrp2 promoters were mapped to RXRalpha:RARalpha-response elements. Gel mobility shift assays demonstrated specific binding of RXRalpha:RARalpha heterodimers to the ntcp and mrp2 retinoid-response elements. The RXRalpha:RARalpha complex was down-regulated by IL-1beta in HepG2 cells. An unexpected finding was that an adjacent CAAT-enhancer-binding protein element was required for maximal transactivation of the ntcp promoter by RXRalpha:RARalpha. Taken together, these studies demonstrate regulation of two hepatobiliary transporter genes by RXRalpha:RARalpha and describe a mechanism which likely contributes to their down-regulation during inflammation.

Albumins↗

Integrative Genotyping and Analysis of Canine Structural Variation Using Long-read and Short-read Data.

Structural variation makes an important contribution to canine evolution and phenotypic differences. Although recent advances in long-read sequencing have enabled the generation of multiple canine genome assemblies, most prior analyses of structural variation have relied on short-read sequencing. To offer a more complete assessment of structural variation in canines, we performed an integrative analysis of structural variants present in 12 canine samples with available long-read and short-read sequencing data along with genome assemblies. Use of long-reads permits the discovery of heterozygous variation that is absent in existing haploid assembly representations while offering a marked increase in the ability to identify insertion variants relative to short-read approaches. Examination of the size spectrum of structural variants shows that dimorphic LINE-1 and SINE variants account for over 45% of all deletions and identified 1,410 LINE-1s with intact open reading frames that show presence-absence dimorphism. Using a graph-based approach, we genotype newly discovered structural variants in an existing collection of 1,879 resequenced dogs and wolves, generating a variant catalog containing a 56.5% increase in the number of deletions and 705% increase in the number of insertions previously found in the analyzed samples. Examination of allele frequencies across admixture components present across breed clades identified 283 structural variants evolving with a signature of selection.

Animals↗

Analysis of the hypervariable region of the Salmonella enterica genome associated with tRNA(leuX).

The divergence of Salmonella enterica and Escherichia coli is estimated to have occurred approximately 140 million years ago. Despite this evolutionary distance, the genomes of these two species still share extensive synteny and homology. However, there are significant differences between the two species in terms of genes putatively acquired via various horizontal transfer events. Here we report on the composition and distribution across the Salmonella genus of a chromosomal region designated SPI-10 in Salmonella enterica serovar Typhi and located adjacent to tRNA(leuX). We find that across the Salmonella genus the tRNA(leuX) region is a hypervariable hot spot for horizontal gene transfer; different isolates from the same S. enterica serovar can exhibit significant variation in this region. Many P4 phage, plasmid, and transposable element-associated genes are found adjacent to tRNA(leuX) in both Salmonella and E. coli, suggesting that these mobile genetic elements have played a major role in driving the variability of this region.

Bacteriophages↗

McClintock's controlling elements: the full story.

The chapter describes the early part of Barbara McClintock's work on DNA transposons in maize, in which she uncovered the Ac-Ds family of mobile "controlling elements". As a basis to understanding the work there is a preamble on the reproductive biology of maize, which explains the particular qualities of the maize plant as an experimental organism for genetics. An account is then given of the cytology of the system that was used to generate intact chromosomes having "sticky" (broken) ends, which is the starting point of the story. Cytogenetic aspects of the chromatid and chromosome breakage-fusion-bridge cycles, deriving from breakage, are then described; this leads on to a discussion about the way in which variegation phenotypes of the maize kernels can be "read" in terms of chromosome breakage. The "genetic earthquake" event of 1944, triggered by introducing broken chromosomes into a zygote from both parents, is then described; and the sequence of events leading to the discovery of Ds and Ac is traced. Finding mobility of Ds from one chromosomal location to another was pure serendipity, and the transposition showed itself while experiments were being undertaken to accurately map Ds. A similar chance observation revealed transposition of Ac as well, and then the relationship between the two elements was elucidated in terms of their autonomous and non-autonomous nature. The chapter concludes with a brief reference to the molecular cloning of Ac and Ds.

Cell Cycle↗